The invention relates generally to animal model systems useful for examining and manipulating neurobehaviors mediated by nicotine. More specifically, the invention relates to knock-in mice having a leucine-to-serine mutation of the xcex14 nicotinic receptor subunit gene resulting in nicotine hypersensitivity, and to methods of using the knock-in mice to identify agents that modulate nicotine addiction and other neurobehaviors.
The mechanism leading from nicotine intake to addiction begins with the activation of neuronal nicotinic acetylcholine receptors (nAChR). Nicotine elicits dopamine release in several regions of the brain, leading to reward, motor learning, and addictive effects. The highest-affinity and most abundant nicotine binding in the brain corresponds to a nAChR formed by xcex14 and xcexc2 subunits. The xcex14 subunit is the principal partner for the xcex22 subunit in brain; xcex22-containing receptors play an important role in nicotine self-administration, in nicotine-stimulated electrophysiological responses in midbrain neurons, and in nicotine-stimulated dopamine release in the ventral striatum. The xcex14 subunit is localized in dopaminergic neurons with tyrosine hydroxylase. The xcex14 and xcex22 subunits are also the site of at least five point mutations that cause the human disease, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE).
Nicotine is known to reduce anxiety, or to produce a bimodal effect on anxiety. Nicotinic receptors modulate the release of neurotransmitters, for example xcex3-aminobutyric acid, dopamine, and serotonin, that have critical roles in the regulation of anxiety. The mechanism by which nicotine reduces anxiety is not well understood, but results from several studies suggest that nicotine-mediated effects on neurons in which nicotine is a co-transmitter may play important roles. Accordingly, a model system for studying nicotinic neurotransmission and the role of the nicotinic acetylcholine receptor is of key importance.
In one embodiment of the invention there is provided a transgenic non-human animal having a transgene comprising a leucine-to-serine mutation of the xcex14 nicotinic receptor subunit chromosomally integrated into germ cells of the animal. The leucine to serine mutation is located in the M2 transmembrane region of the acetylcholine receptor.
In another embodiment of the invention there is provided a transgenic mouse comprising a transgene having a leucine-to-serine mutation at 9xe2x80x2 of the xcex14 nicotinic receptor subunit. Expression of the receptor subunit gene results in a mouse that displays modified behavior compared to a normal mouse. The transgenic mouse displays nicotinic hypersensitivity, increased anxiety, increased sensitivity to seizures, poor motor learning, excessive ambulation, a reduction in dopaminergic neuron function upon aging, or any combination thereof.
In another embodiment of the invention there is provided a transgenic mouse comprising a transgene having a single codon change in the xcex14 nicotinic receptor subunit. Expression of the receptor subunit gene results in a mouse that displays modified behavior compared to a normal mouse. The transgenic mouse displays nicotinic hypersensitivity, increased anxiety, increased sensitivity to seizures, poor motor learning, excessive ambulation, a reduction in dopaminergic neuron function upon aging, susceptibility to seizure, spontaneous seizures, or any combination thereof.
In yet another embodiment of the invention there is provided a method for screening a candidate agent for the ability to modulate nicotine-mediated behavior in a transgenic animal. The method includes administering to a first transgenic animal a candidate agent and comparing nicotine-mediated behavior in the animal to the nicotine-mediated behavior of a second transgenic animal not administered the candidate agent. A difference in nicotine-mediated behavior in the animal administered the candidate agent compared to the animal not administered the agent is indicative of an agent that modifies nicotine-mediated behavior.
In still another embodiment of the invention there is provided a method for screening for candidate agents that modulate nicotine hypersensitivity. The method includes administering a candidate agent to a transgenic animal and determining the effect of the agent upon a cellular or molecular process associated with nicotinic hypersensitivity compared to an effect of the agent administered to a non-transgenic animal.
In another embodiment, there is provided a method for screening for candidate agents that modulate seizures associated with epilepsy. The method includes administering a candidate agent to a transgenic animal and determining the effect of the agent upon seizure activity associated with epilepsy compared to an effect of the agent administered to a non-transgenic animal.
FIG. 1 shows the physiological design, recombinant construction, and genomic characterization of the xcex14 knock-in mouse strains.
FIGS. 1A-C show the agonist concentration-response relations of WT and mutated (xcex14 Leu9xe2x80x2Ser) rat xcex14xcex22 receptors expressed in oocytes (five oocytes for each curve).
In FIG. 1A, the agonist is acetylcholine; in
FIG. 1B, the agonist is nicotine and in
FIG. 1C, the agonist is choline. The choline responses of the WT receptor were not studied systematically, because there is no response at choline concentrations up to 1 mM, and higher concentrations of choline block the channel. FIG. 1C insert shows the time course of the response to 30 xcexcM choline, showing partial desensitization.
FIG. 1D shows the targeting construct containing exon 5 with the Leu9xe2x80x2Ser mutation, the neomycin resistance gene (neo) flanked by loxP sites, the diphtheria toxin A chain gene (DT), and the pKO V907 vector (pKO).
FIG. 1E shows that deletion of the neo cassette by transfecting the neo-intact ES cells with a cytomegalovirus-Cre plasmid generates neo-deleted ES cell lines.
FIG. 1F shows sequence analysis of DNA extracted from WT (SEQ ID NO:2) heterozygous (het: SEQ ID NO:3), and homozygous (hom: SEQ ID NO:5) neo-intact mice. The WT sequence at nucleotide position 142, corresponding to the codon at position 9xe2x80x2 in the M2 region, is CTT, encoding leucine; the mutant sequence is TCT, encoding serine.
FIG. 2 shows the pathophysiological basis of dopaminergic neuron deficits in mutant mice.
FIG. 2A shows cell counts of tyrosine hydroxylase (TH)-positive neurons in substantia nigra of ED 16 to ED 18 embryos from WT, neo-intact, and neo-deleted mice. The heterozygote (het) cell counts do not differ significantly from WT, but both the homozygous (homo) neo-intact (P, 0.01, f test) and the neo-deleted cell counts (P, 0.05, t test) differ significantly from WT.
FIG. 2B shows whole-cell voltage-clamp recording of responses to two consecutive puffs of choline (100 xcexcM, 20 ms) in neuron-like cells differentiated from ED 16 midbrain neuronal progenitor cells. Upper trace, cell from a WT embryo; lower trace, cell derived from a heterozygous neo-intact ED 16 embryo.
FIG. 2C shows the mean xc2x1SEM of responses in neuron-like cells derived from heterozygous animals (n=5 cells) but little or no response in cells from WT animals (n=7 cells; significant difference, P, 0.05, t test).
FIG. 3 shows spontaneous and drug-modulated locomotion of WT and heterozygous (het) neo-intact mutants.
FIG. 3A shows the effect of no treatment. Heterozygotes showed significantly higher locomotion than WT mice at the beginning of the experiment (P, 0.001).
FIG. 3B shows locomotion after nicotine, 0.02 mg/kg, was injected 30 min after the start of behavioral monitoring. The plot shows data averaged over the time periods, 10 min before baseline (BL) and 5-15 min after injection. Heterozygous mice showed a significant reduction of locomotor activity after nicotine injection (P, 0.05). There was no significant difference in non-injected animals (right-hand bars), nicotine-injected control animals, or saline-injected WT or heterozygous animals.
FIG. 3C shows that heterozygotes were impaired compared with WTs on the accelerating rotarod (P, 0.012) when tested for three sequential days (n=20xe2x88x9222 of each genotype).
FIGS. 3D and 3E show the effect of amphetamine on locomotion of WT and heterozygous (het) mice at two ages. Before drug administration, animals were allowed to habituate for 30 min. Ten male WT and 10 male heterozygous mice showing at least a three-fold increased activity over baseline in response to amphetamine at three months of age were selected for longitudinal follow-up studies.
FIG. 3D shows a comparison in amphetamine responses for heterozygotes at 3 months vs. 11 months of age.
FIG. 3E shows the average activity plotted for the period between 5 and 45 min after injection. The response at 11 months declines significantly compared with the response at three months in heterozygous mice [F(1,9)=12.72, P, 0.01] but not in WT mice.
FIG. 4 shows the increased anxiety in xcex14 heterozygotes (het) compared with WT mice in the elevated plus maze (A) and mirrored chamber (B)(n=22 mice of each genotype).
FIG. 4A shows that heterozygotes were significantly more anxious, as measured by percentage of entrances into the open arms (P, 0.01), percentage of time in the open arms (P, 0.002), percentage of time in the closed arms (P, 0.02), and entrances to the end of the open arms (P, 0.005).
FIG. 4B shows that heterozygotes were significantly more anxious in the mirrored chamber, as measured by latency to enter the mirrored chamber (P, 0.026), percentage of time in the mirrored chamber (P, 0.03), entrances into the mirrored chamber (P, 0.02), but not by the number of entries into the mirrored passage.
FIG. 5 shows behavioral scoring of seizure activity in heterozygous and wild type mice following subcutaneous nicotine injection.
FIG. 5A shows seizure scores and
FIG. 5B shows Straub tail scores. The means of six animals xc2x11 SE are shown.